Available online at www.sciencedirect.com
ScienceDirect IFITM proteins — cellular inhibitors of viral entry SE Smith1, S Weston2, P Kellam1,3 and M Marsh2 Interferon inducible transmembrane (IFITM) proteins are a recently discovered family of cellular anti-viral proteins that restrict the replication of a number of enveloped and nonenveloped viruses. IFITM proteins are located in the plasma membrane and endosomal membranes, the main portals of entry for many viruses. Biochemical and membrane fusion studies suggest IFITM proteins have the ability to inhibit viral entry, possibly by modulating the fluidity of cellular membranes. Here we discuss the IFITM proteins, recent work on their mode of action, and future directions for research. Addresses 1 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK 2 MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK 3 MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, Gower Street, London WC1E 6BT, UK Corresponding authors: Kellam, P ([email protected]) and Marsh, M ([email protected])
Current Opinion in Virology 2014, 4:71–77 This review comes from a themed issue on Virus entry Edited by Mark Marsh and Jane A McKeating For a complete overview see the Issue and the Editorial Available online 28th January 2014 1879-6257/$ – see front matter, # 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2013.11.004
Introduction In recent years, studies of innate defence mechanisms have identified a number of cellular proteins that interfere with the replication of human and animal viruses. Many of these so-called ‘restriction factors’ have been most intensively studied for human immunodeficiency virus (HIV-1). For example, tripartite motif-containing protein 5 (TRIM5) [1], APOBEC3G [2], 20 ,30 -cyclicnucleotide 30 -phosphodiesterase [3] and tetherin [4] have been found to affect uncoating, reverse transcription, virus assembly and virus release, respectively. A new addition to this antiviral repertoire is myxoma resistance protein B (MxB/Mx2) [5] that inhibits HIV-1 at a late post-entry step. However, restriction factors for other viruses have also been identified, including: RNA-activated protein kinase (PKR), that restricts Hepatitis C and other viruses [6]; MX1, that restricts influenza A virus (IAV) and measles virus [7]; and 20 -50 -oligoadenylate synthase/RNase L, that restricts Hepatitis C and other viruses [8]. Many of these factors are components of the www.sciencedirect.com
broad antiviral response induced by interferons, collectively known as interferon stimulated genes (ISGs: for review see [9]). Although recognised to act at different stages in viral replication cycles, most of the well-characterised restriction factors affect steps following virus entry. Recently, a new family of proteins has been identified that appears to act specifically on virus entry, the interferon inducible transmembrane (IFITM) proteins. Here we review the antiviral capacity of three of these proteins, IFITM1–3.
Identification of IFITMs The IFITM gene family was initially identified more than 20 years ago [10], with particular interest in the interferon-stimulated response elements (ISREs) they contained. The IFITM transcripts were originally named 9-27, 1-8D and 1-8U, however, the antiviral properties of the encoded proteins were only identified in 2009 in an RNAi screen for host factors that influence IAV replication. Knock-down of IFITM3 led to enhanced viral replication. Conversely, overexpression of IFITM1, 2, or 3 inhibited early viral replication [11]. Subsequent genome analyses have indicated that the IFITM genes are likely to have arisen by gene duplication very early in vertebrate evolution [12], since ‘lower’ vertebrates, such as lampreys, possess at least one IFITM-like gene [13]. To date, five IFITM genes have been identified in humans, of which IFITM1, 2, 3 and 5 are clustered within a 26 kb region towards the telomere on the short arm of chromosome 11. IFITM5 is not IFN inducible and is involved in bone mineralisation [14]. The fifth gene, IFITM10, is located 1.4 Mb towards the centromere, but little is known about its function. IFITM4 is not present in humans, but is located close to Ifitm1, 2, 3, and 5 in the mouse genome [15], in which the locus has expanded to encode seven Ifitm genes. Analogous genes have also been found in other mammals [12], including marsupials [13], and avian species [16]. Although the molecular function of these proteins has been largely studied in cell culture systems, studies in mice and humans suggest IFITM proteins, and IFITM3 in particular, restrict IAV infection in vivo. Ifitm3/ mice fail to control infection by mildly-pathogenic strains of IAV compared to their wild type littermates, developing fatal fulminant viral pneumonia [17,18]. Everitt et al. also found that the minor C allele of human IFITM3 (synonymous single nucleotide polymorphism (SNP) rs12252) was enriched in a cohort of Caucasian patients Current Opinion in Virology 2014, 4:71–77
72 Virus entry
hospitalised with either IAV H1N1/09 or influenza B in the 2009 pandemic [17]. Although the C allele is rare in Caucasians, replication of this genetic association was shown in a cohort of Han Chinese patients (the SNP is more prevalent in this population) with severe symptoms following influenza infection. The minority CC genotype was found in 69% of patients with severe disease compared to only 25% with mild symptoms [19], further suggesting that deleterious changes in the IFITM3 gene can influence the severity of influenza infection. It is currently unclear how this allele impacts IAV pathogenesis, but the alteration of a splice acceptor site may lead to the synthesis of a truncated IFITM3 protein that lacks the N-terminal 21 amino acids, and is expressed primarily on the cell surface rather than in endosomes (see below) [17,20]. Aside from rs12252, little investigation has been carried out into other SNPs reported for IFITM3. One study carried out by John et al. [21] made alterations of non-synonymous SNPs H3Q/rs1136853, D56G/rs55794999, H57D/rs1553883,
N69D/rs12778, and G95R/rs61744108, with only G95R showing a small reduction in IAV restriction compared to wild type.
Broad-spectrum antiviral function Using cell culture systems, and often pseudotype viruses, several groups demonstrated that, in addition to IAV, entry and infection by representatives of multiple virus families (including filoviruses, rhabdoviruses and flaviviruses) [22,23,24] were also inhibited by overexpression of IFITMs, particularly IFITM3 (see Table 1). Interestingly, these restricted viruses are all enveloped, with ssRNA genomes, and considered to enter cells by membrane fusion following endocytosis. However, some retroviruses (e.g. Moloney leukaemia virus (MLV)) and several arenaviruses were apparently not restricted. Although restriction of HIV-1 infection was not initially detected [11], several more recent studies have reported some restriction of cell infection [20,25,26]. Most
Table 1 Summary of viruses IFITM proteins have been tested against Virus
pH dependent
Restricts infectivity
Prevents cell–cell fusion
Pseudotyped virions (P) or live virus (L)
Influenza A virus
UU
U
U
PL
M1–3
Influenza B virus West Nile virus Dengue virus Hepatitis C virus
UU U UU U
U U U U/
L P P PL
Vesicular stomatitis virus Rabies virus Lagos Bat virus Marburg virus Ebola virus SARS coronavirus HIV-1
U UU UU D D D
U U U U U U U/
M1–3 M1–3 M1–3 M3 — no, M1 — yes M1–3 M2–3 M2–3 M1–3 M1–3 M1–3 Mixed results
Moloney leukaemia virus
Jaagsiekte sheep retrovirus Lassa virus Machupo virus Lymphocytic choriomeningitis virus Semliki Forest virus
U U U U
U
U
U
La Crosse virus Hantaan virus Andes virus Rift Valley fever virus Crimean–Congo haemorrhagic fever virus
UU UU UU UU UU
U U U U
Reovirus
UU
U
Family
Enveloped Orthomyxoviridae
Flaviviridae
Rhabdoviridae
Filoviridae Coronaviridae Retroviridae
Arenaviridae
Alphaviridae Bunyaviridae
Non-enveloped Reoviridae
U
P P P P P P P
L
L L L L
Restriction status
Reference
Brass et al. [11], Smith et al. [16] Everitt et al. [17] Brass et al. [11] Brass et al. [11] Brass et al. [11], Wilkins et al. [24] Weidner et al. [23] Smith et al. [16] Smith et al. [16] Huang et al. [22] Huang et al. [22] Huang et al. [22] Brass et al. [11], Lu et al. [26], Jia et al. [20] Brass et al. [11], Huang et al. [22] Li et al. [28] Brass et al. [11] Brass et al. [11] Brass et al. [11]
PL
No
U
P P P P
M1 best No No No
U
L L L L L-attenuated L
M2 and M3 best M1-3 M1-3 M1-3 M2 and M3 No
Mudhasani Mudhasani Mudhasani Mudhasani Mudhasani
L
M3
Anafu et al. [27]
Li et al. [28] et et et et et
al. al. al. al. al.
[30] [30] [30] [30] [30]
U, fuses at pH >6; UU, fuses at pH
ScienceDirect IFITM proteins — cellular inhibitors of viral entry SE Smith1, S Weston2, P Kellam1,3 and M Marsh2 Interferon inducible transmembrane (IFITM) proteins are a recently discovered family of cellular anti-viral proteins that restrict the replication of a number of enveloped and nonenveloped viruses. IFITM proteins are located in the plasma membrane and endosomal membranes, the main portals of entry for many viruses. Biochemical and membrane fusion studies suggest IFITM proteins have the ability to inhibit viral entry, possibly by modulating the fluidity of cellular membranes. Here we discuss the IFITM proteins, recent work on their mode of action, and future directions for research. Addresses 1 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK 2 MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK 3 MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, Gower Street, London WC1E 6BT, UK Corresponding authors: Kellam, P ([email protected]) and Marsh, M ([email protected])
Current Opinion in Virology 2014, 4:71–77 This review comes from a themed issue on Virus entry Edited by Mark Marsh and Jane A McKeating For a complete overview see the Issue and the Editorial Available online 28th January 2014 1879-6257/$ – see front matter, # 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2013.11.004
Introduction In recent years, studies of innate defence mechanisms have identified a number of cellular proteins that interfere with the replication of human and animal viruses. Many of these so-called ‘restriction factors’ have been most intensively studied for human immunodeficiency virus (HIV-1). For example, tripartite motif-containing protein 5 (TRIM5) [1], APOBEC3G [2], 20 ,30 -cyclicnucleotide 30 -phosphodiesterase [3] and tetherin [4] have been found to affect uncoating, reverse transcription, virus assembly and virus release, respectively. A new addition to this antiviral repertoire is myxoma resistance protein B (MxB/Mx2) [5] that inhibits HIV-1 at a late post-entry step. However, restriction factors for other viruses have also been identified, including: RNA-activated protein kinase (PKR), that restricts Hepatitis C and other viruses [6]; MX1, that restricts influenza A virus (IAV) and measles virus [7]; and 20 -50 -oligoadenylate synthase/RNase L, that restricts Hepatitis C and other viruses [8]. Many of these factors are components of the www.sciencedirect.com
broad antiviral response induced by interferons, collectively known as interferon stimulated genes (ISGs: for review see [9]). Although recognised to act at different stages in viral replication cycles, most of the well-characterised restriction factors affect steps following virus entry. Recently, a new family of proteins has been identified that appears to act specifically on virus entry, the interferon inducible transmembrane (IFITM) proteins. Here we review the antiviral capacity of three of these proteins, IFITM1–3.
Identification of IFITMs The IFITM gene family was initially identified more than 20 years ago [10], with particular interest in the interferon-stimulated response elements (ISREs) they contained. The IFITM transcripts were originally named 9-27, 1-8D and 1-8U, however, the antiviral properties of the encoded proteins were only identified in 2009 in an RNAi screen for host factors that influence IAV replication. Knock-down of IFITM3 led to enhanced viral replication. Conversely, overexpression of IFITM1, 2, or 3 inhibited early viral replication [11]. Subsequent genome analyses have indicated that the IFITM genes are likely to have arisen by gene duplication very early in vertebrate evolution [12], since ‘lower’ vertebrates, such as lampreys, possess at least one IFITM-like gene [13]. To date, five IFITM genes have been identified in humans, of which IFITM1, 2, 3 and 5 are clustered within a 26 kb region towards the telomere on the short arm of chromosome 11. IFITM5 is not IFN inducible and is involved in bone mineralisation [14]. The fifth gene, IFITM10, is located 1.4 Mb towards the centromere, but little is known about its function. IFITM4 is not present in humans, but is located close to Ifitm1, 2, 3, and 5 in the mouse genome [15], in which the locus has expanded to encode seven Ifitm genes. Analogous genes have also been found in other mammals [12], including marsupials [13], and avian species [16]. Although the molecular function of these proteins has been largely studied in cell culture systems, studies in mice and humans suggest IFITM proteins, and IFITM3 in particular, restrict IAV infection in vivo. Ifitm3/ mice fail to control infection by mildly-pathogenic strains of IAV compared to their wild type littermates, developing fatal fulminant viral pneumonia [17,18]. Everitt et al. also found that the minor C allele of human IFITM3 (synonymous single nucleotide polymorphism (SNP) rs12252) was enriched in a cohort of Caucasian patients Current Opinion in Virology 2014, 4:71–77
72 Virus entry
hospitalised with either IAV H1N1/09 or influenza B in the 2009 pandemic [17]. Although the C allele is rare in Caucasians, replication of this genetic association was shown in a cohort of Han Chinese patients (the SNP is more prevalent in this population) with severe symptoms following influenza infection. The minority CC genotype was found in 69% of patients with severe disease compared to only 25% with mild symptoms [19], further suggesting that deleterious changes in the IFITM3 gene can influence the severity of influenza infection. It is currently unclear how this allele impacts IAV pathogenesis, but the alteration of a splice acceptor site may lead to the synthesis of a truncated IFITM3 protein that lacks the N-terminal 21 amino acids, and is expressed primarily on the cell surface rather than in endosomes (see below) [17,20]. Aside from rs12252, little investigation has been carried out into other SNPs reported for IFITM3. One study carried out by John et al. [21] made alterations of non-synonymous SNPs H3Q/rs1136853, D56G/rs55794999, H57D/rs1553883,
N69D/rs12778, and G95R/rs61744108, with only G95R showing a small reduction in IAV restriction compared to wild type.
Broad-spectrum antiviral function Using cell culture systems, and often pseudotype viruses, several groups demonstrated that, in addition to IAV, entry and infection by representatives of multiple virus families (including filoviruses, rhabdoviruses and flaviviruses) [22,23,24] were also inhibited by overexpression of IFITMs, particularly IFITM3 (see Table 1). Interestingly, these restricted viruses are all enveloped, with ssRNA genomes, and considered to enter cells by membrane fusion following endocytosis. However, some retroviruses (e.g. Moloney leukaemia virus (MLV)) and several arenaviruses were apparently not restricted. Although restriction of HIV-1 infection was not initially detected [11], several more recent studies have reported some restriction of cell infection [20,25,26]. Most
Table 1 Summary of viruses IFITM proteins have been tested against Virus
pH dependent
Restricts infectivity
Prevents cell–cell fusion
Pseudotyped virions (P) or live virus (L)
Influenza A virus
UU
U
U
PL
M1–3
Influenza B virus West Nile virus Dengue virus Hepatitis C virus
UU U UU U
U U U U/
L P P PL
Vesicular stomatitis virus Rabies virus Lagos Bat virus Marburg virus Ebola virus SARS coronavirus HIV-1
U UU UU D D D
U U U U U U U/
M1–3 M1–3 M1–3 M3 — no, M1 — yes M1–3 M2–3 M2–3 M1–3 M1–3 M1–3 Mixed results
Moloney leukaemia virus
Jaagsiekte sheep retrovirus Lassa virus Machupo virus Lymphocytic choriomeningitis virus Semliki Forest virus
U U U U
U
U
U
La Crosse virus Hantaan virus Andes virus Rift Valley fever virus Crimean–Congo haemorrhagic fever virus
UU UU UU UU UU
U U U U
Reovirus
UU
U
Family
Enveloped Orthomyxoviridae
Flaviviridae
Rhabdoviridae
Filoviridae Coronaviridae Retroviridae
Arenaviridae
Alphaviridae Bunyaviridae
Non-enveloped Reoviridae
U
P P P P P P P
L
L L L L
Restriction status
Reference
Brass et al. [11], Smith et al. [16] Everitt et al. [17] Brass et al. [11] Brass et al. [11] Brass et al. [11], Wilkins et al. [24] Weidner et al. [23] Smith et al. [16] Smith et al. [16] Huang et al. [22] Huang et al. [22] Huang et al. [22] Brass et al. [11], Lu et al. [26], Jia et al. [20] Brass et al. [11], Huang et al. [22] Li et al. [28] Brass et al. [11] Brass et al. [11] Brass et al. [11]
PL
No
U
P P P P
M1 best No No No
U
L L L L L-attenuated L
M2 and M3 best M1-3 M1-3 M1-3 M2 and M3 No
Mudhasani Mudhasani Mudhasani Mudhasani Mudhasani
L
M3
Anafu et al. [27]
Li et al. [28] et et et et et
al. al. al. al. al.
[30] [30] [30] [30] [30]
U, fuses at pH >6; UU, fuses at pH
